RESUMEN
RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.
Asunto(s)
Ribonucleoproteína Heterogénea-Nuclear Grupo M , Intrones , Elementos de Nucleótido Esparcido Largo , Empalme del ARN , ARN Bicatenario , Humanos , Ribonucleoproteína Heterogénea-Nuclear Grupo M/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo M/metabolismo , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Elementos de Nucleótido Esparcido Largo/genética , Interferones/metabolismo , Interferones/genética , Animales , Células HEK293 , Ratones , Transcriptoma , Exones , Sitios de Empalme de ARN , Elementos Alu/genéticaRESUMEN
Gene Ontology (GO) is widely used in the biological domain. It is the most comprehensive ontology providing formal representation of gene functions (GO concepts) and relations between them. However, unintentional quality defects (e.g. missing or erroneous relations) in GO may exist due to the large size of GO concepts and complexity of GO structures. Such quality defects would impact the results of GO-based analyses and applications. In this work, we introduce a novel evidence-based lexical pattern approach for quality assurance of GO relations. We leverage two layers of evidence to suggest potentially missing relations in GO as follows. We first utilize related concept pairs (i.e. existing relations) in GO to extract relationship-specific lexical patterns, which serve as the first layer evidence to automatically suggest potentially missing relations between unrelated concept pairs. For each suggested missing relation, we further identify two other existing relations as the second layer of evidence that resemble the difference between the missing relation and the existing relation based on which the missing relation is suggested. Applied to the 15 December 2021 release of GO, this approach suggested a total of 866 potentially missing relations. Local domain experts evaluated the entire set of potentially missing relations, and identified 821 as missing relations and 45 indicate erroneous existing relations. We submitted these findings to the GO consortium for further validation and received encouraging feedback. These indicate that our evidence-based approach can be utilized to uncover missing relations and erroneous existing relations in GO.
Asunto(s)
Ontología de GenesRESUMEN
BACKGROUND: Skin scarring can occur after punch biopsies, prohibiting their routine utilization, especially in the central face. OBJECTIVES: This paper describes a scarless, 0.33-mm-diameter skin microbiopsy for molecular analysis of skin. METHODS: This is was single-center, randomized, prospective study with 15 patients receiving no biopsy or biopsy on the left or right nasolabial fold. Six blinded raters assessed participant photos at baseline, 1 month, and 3 months post biopsy to evaluate for a visualized scar. Patient and Observer Scar Assessment Scale was completed. Additionally, biopsies from various skin regions of body along with arm skin after treatment with a single Erbium-YAG laser were processed for molecular analysis. RESULTS: No patients exhibited scar formation based on evaluation of photographs and patient feedback. There was no mark at the biopsy site 7 days post-procedure. Optical coherence tomography showed a complete closing of the biopsy-punch wound 48 hours post-biopsy. One month post-biopsy, photography reviewers were unable to identify a scar, on average, 90% of the time at 3-month follow-up. Microbiopsies from various anatomical regions were successfully extracted for histology, electron microscopy, and gene expression analysis. Selected skin rejuvenation markers in the biopsies from Erbium-YAG-treated forearm skin resulted in significant gene upregulation in extracellular matrix molecules at 1 month posttreatment compared with untreated skin. CONCLUSIONS: A core microbiopsy of 0.33 mm can be extracted reproducibly for histological, ultrastructural, and gene expression analysis without scarring. This allows repeated sampling for assessment of skin treatments and diseases, including aesthetics and wound-healing progress.
Asunto(s)
Terapia por Láser , Piel , Cicatriz , Humanos , Surco Nasolabial/patología , Estudios Prospectivos , Piel/patología , Cicatrización de HeridasRESUMEN
BACKGROUND: Multiple processing and handling methods of autologous fat yield to variations in graft retention and viability, which results in unpredictable clinical outcomes. OBJECTIVES: This study aims to understand the skin effects of fat graft preparations that contain a varying ratio of free-lipid and stem-cell-bearing stromal vascular fractions (SVF). METHODS: Lipoaspirates from consenting patients were processed into emulsified fat and then SVF and adipocyte fractions (free-lipid). SVF enriched with 0%, 5%, and 15% free-lipid were grafted along the dorsum of athymic rats. The xenografts were collected 45 days after grafting and then prepped for immunostaining. RESULTS: Xenografts resulted in viable tissue mass under the panniculus carnosus of rats as confirmed with human specific markers. A low percentage of human cells was also detected in the lower reticular dermis. Although grafts with SVF formed adipocytes of normal architecture, grafts formed with free-lipid alone resulted in large lipid vacuoles in varying sizes. Among graft preparations, SVF with 10% free-lipid resulted in much-developed adipocyte architecture with collagen and elastin. Compared with SVF alone grafts, SVF with free-lipid had higher CD44 expression, suggesting a localized immune response of adipocytes. CONCLUSIONS: Current studies suggest that SVF enriched with approximately 10% free-lipid provides the best conditions for fat graft differentiation into viable fat tissue formation as well as collagen and elastin production to provide mechanical support for overlaying skin in an athymic rat model. Additionally, application of this therapeutic modality in a simple clinical setting may offer a practical way to concentrate SVF with free-lipid in a small volume for the improvement of clinical defects.
Asunto(s)
Tejido Adiposo/trasplante , Diferenciación Celular , Supervivencia de Injerto/fisiología , Adipocitos/fisiología , Tejido Adiposo/citología , Adulto , Animales , Separación Celular , Femenino , Humanos , Lípidos/fisiología , Masculino , Células Madre Mesenquimatosas/fisiología , Persona de Mediana Edad , Ratas , Ratas Desnudas , Trasplante Autólogo/métodos , Trasplante Heterólogo/métodosRESUMEN
PURPOSE: Cisplatin (CDDP)-based chemotherapy is a first-line treatment for patients with advanced head and neck squamous cell carcinomas (HNSCC), despite a high rate of treatment failures, acquired resistance, and subsequent aggressive behavior. The purpose of this study was to study the mechanism of CDDP resistance and metastasis in HNSCC. We investigated the role of NRF2 pathway activation as a driven event for tumor progression and metastasis of HNSCC. EXPERIMENTAL DESIGN: Human HNSCC cell lines that are highly resistant to CDDP were generated. Clonogenic survival assays and a mouse model of oral cancer were used to examine the impact of NRF2 activation in vitro and in vivo on CDDP sensitivity and development of metastasis. Western blotting, immunostaining, whole-exome sequencing, single-cell transcriptomic and epigenomic profiling platforms were performed to dissect clonal evolution and molecular mechanisms. RESULTS: Implantation of CDDP-resistant HNSCC cells into the tongues of nude mice resulted in a very high rate of distant metastases. The CDDP-resistant cells had significantly higher expression of NRF2 pathway genes in the presence of newly acquired KEAP1 mutations, or via epigenomic activation of target genes. Knockdown of NRF2 or restoration of the wild-type KEAP1 genes resensitized resistant cells to CDDP and decreased distant metastasis (DM). Finally, treatment with inhibitor of glutaminase-1, a NRF2 target gene, alleviated CDDP resistance. CONCLUSIONS: CDDP resistance and development of DM are associated with dysregulated and epigenetically reprogrammed KEAP1-NRF2 signaling pathway. A strategy targeting KEAP1/NRF2 pathway or glutamine metabolism deserves further clinical investigation in patients with CDDP-resistant head and neck tumors.
Asunto(s)
Antineoplásicos , Neoplasias de Cabeza y Cuello , Factor 2 Relacionado con NF-E2 , Carcinoma de Células Escamosas de Cabeza y Cuello , Animales , Humanos , Ratones , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Cisplatino/farmacología , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos/genética , Epigénesis Genética , Epigenómica , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/genética , Proteína 1 Asociada A ECH Tipo Kelch/genética , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Ratones Desnudos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/genéticaRESUMEN
Despite advances in surgery, chemotherapy, and radiation, there are limited treatment options for advanced head and neck squamous cell carcinoma (HNSCC) and survival remains very poor. Therefore, effective therapies are desperately needed. Recently, selective exploitation of DNA damage and replication stress responses has become a novel approach for cancer treatment. Wee1 kinase and Rad51 recombinase are two proteins involved in regulating replication stress and homologous recombination repair in cancer cells. In this study, we investigated the combined effect of Rad51 inhibitor (B02) and Wee1 inhibitor (AZD1775) in vitro and in vivo in various HNSCC cell lines. Clonogenic survival assays demonstrated that B02 synergized with AZD1775 in vitro in all HNSCC cell lines tested. The synergy between these drugs was associated with forced CDK1 activation and reduced Chk1 phosphorylation leading to induction of excessive DNA damage and replication stress, culminating in aberrant mitosis and apoptosis. Our results showed that elevated Rad51 mRNA expression correlated with worse survival in HNSCC patients with HPV-positive tumors. The combination of B02 and AZD1775 significantly inhibited tumor growth in vivo in mice bearing HPV-positive HNSCC tumors as compared to HPV-negative HNSCC. This differential sensitivity appears to be linked to HPV-positive tumors having more in vivo endogenous replication stress owing to transformation by E6 and E7 oncogenes. Furthermore, addition of B02 radiosensitized the HPV-negative HNSCC tumors in vitro and in vivo In conclusion, our data implicate that a novel rational combination with Rad51 and Wee1 inhibitors holds promise as synthetic lethal therapy, particularly in high-risk HPV-positive HNSCC.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Daño del ADN/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Recombinasa Rad51/antagonistas & inhibidores , Animales , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Biología Computacional/métodos , Reparación del ADN/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Perfilación de la Expresión Génica , Recombinación Homóloga , Humanos , Ratones , Pirazoles/farmacología , Pirimidinonas/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Carcinoma de Células Escamosas de Cabeza y Cuello/tratamiento farmacológico , Carcinoma de Células Escamosas de Cabeza y Cuello/etiología , Carcinoma de Células Escamosas de Cabeza y Cuello/metabolismo , Carcinoma de Células Escamosas de Cabeza y Cuello/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Plasma hyaluronan (HA) increases systemically in type 2 diabetes (T2D) and the HA synthesis inhibitor, 4-Methylumbelliferone, has been proposed to treat the disease. However, HA is also implicated in normal physiology. Therefore, we generated a Hyaluronan Synthase 2 transgenic mouse line, driven by a tet-response element promoter to understand the role of HA in systemic metabolism. To our surprise, adipocyte-specific overproduction of HA leads to smaller adipocytes and protects mice from high-fat-high-sucrose-diet-induced obesity and glucose intolerance. Adipocytes also have more free glycerol that can be released upon beta3 adrenergic stimulation. Improvements in glucose tolerance were not linked to increased plasma HA. Instead, an HA-driven systemic substrate redistribution and adipose tissue-liver crosstalk contributes to the systemic glucose improvements. In summary, we demonstrate an unexpected improvement in glucose metabolism as a consequence of HA overproduction in adipose tissue, which argues against the use of systemic HA synthesis inhibitors to treat obesity and T2D.